Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Hum Reprod. 2010 Jul;25(7):1643-56. doi: 10.1093/humrep/deq105. Epub 2010 May 26.
It has been suggested that the zona pellucida (ZP) may mediate species-specific fertilization. In human the ZP is composed of four glycoproteins: ZP1, ZP2, ZP3 and ZP4. In the present study, the expression profile of ZP1 in human oocytes and ovaries, and its role during fertilization, is presented.
Human ZP1 (amino acid residues 26-551) was cloned and expressed in both non-glycosylated and glycosylated forms and its ability to bind to the capacitated human spermatozoa and to induce acrosomal exocytosis was studied. Monoclonal antibodies (MAbs), specific for human ZP1 and devoid of reactivity with ZP2, ZP3 and ZP4 were generated and used to localize native ZP1 in oocytes and ovarian tissues.
The MAbs generated against ZP1 recognized specifically the zona matrix of secondary and antral follicles, ovulated oocytes, atretic follicles and degenerating intravascular oocytes, but failed to react with the Fallopian tube, endometrium, ectocervix and kidney. Escherichia coli and baculovirus-expressed recombinant human ZP1 revealed bands of approximately 75 and approximately 85 kDa, respectively, in western blot. Lectin binding studies revealed the presence of both N- and O-linked glycosylation in baculovirus-expressed ZP1. Fluorescein isothiocyanate-labelled E. coli- and baculovirus-expressed recombinant ZP1 bound to the anterior head of capacitated spermatozoa, however, only baculovirus-expressed ZP1 induced acrosomal exocytosis in capacitated sperm suggesting the importance of glycosylation in mediating the acrosome reaction. The human ZP1-mediated acrosome reaction involved the activation of both T- and L-type voltage-operated calcium channels, but does not activate the G(i)-coupled receptor pathway. Inhibition of protein kinase A and C significantly also reduced the ZP1-mediated induction of the acrosome reaction.
These studies revealed for the first time that in humans ZP1, in addition to ZP3 and ZP4, binds to capacitated spermatozoa and induces acrosomal exocytosis.
有人认为透明带（ZP）可能介导种间受精。在人类中，ZP 由四种糖蛋白组成：ZP1、ZP2、ZP3 和 ZP4。本研究介绍了人类卵母细胞和卵巢中 ZP1 的表达谱及其在受精过程中的作用。
克隆并表达了非糖基化和糖基化形式的人 ZP1（氨基酸残基 26-551），研究了其与人获能精子结合的能力以及诱导顶体反应的能力。生成了针对人 ZP1 的单克隆抗体（MAb），这些 MAb 特异性识别 ZP1，而不与 ZP2、ZP3 和 ZP4 反应，并用于定位卵母细胞和卵巢组织中的天然 ZP1。
针对 ZP1 生成的 MAb 特异性识别次级和窦卵泡、排卵卵母细胞、闭锁卵泡和退化的血管内卵母细胞的透明带基质，但与输卵管、子宫内膜、宫颈外口和肾脏不反应。E.coli 和杆状病毒表达的重组人 ZP1 在 Western blot 中分别显示约 75 和约 85 kDa 的条带。凝集素结合研究表明，杆状病毒表达的 ZP1 存在 N 和 O 连接的糖基化。FITC 标记的 E.coli 和杆状病毒表达的重组 ZP1 与获能精子的前头部结合，但只有杆状病毒表达的 ZP1 诱导获能精子发生顶体反应，表明糖基化在介导顶体反应中的重要性。人 ZP1 介导的顶体反应涉及 T 和 L 型电压门控钙通道的激活，但不激活 G(i)偶联受体途径。PKA 和 PKC 的抑制也显著降低了 ZP1 介导的顶体反应的诱导。
这些研究首次表明，在人类中，除了 ZP3 和 ZP4 之外，ZP1 还与获能精子结合并诱导顶体反应。
